Cbl-b, a ring-type E3 ubiquitin protein ligase, is implicated in setting the threshold of T lymphocyte activation. The p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) was identified as a substrate for Cbl-b. We have shown that Cbl-b negatively regulated p85 in a proteolysis-independent manner. Cbl-b is involved in the recruitment of p85 to CD28 and T cell antigen receptor ζ through its E3 ubiquitin ligase activity. The enhanced activation of Cbl-b^−/− T cells was suppressed by the inhibition of PI3K. The results suggest a proteolysis-independent function for Cbl-b in the modification of protein recruitment.